Diselenides as universal oxidative folding catalysts of diverse proteins

Small-molecule diselenides show considerable potential as catalysts of oxidative protein folding. To explore their scope, diselenide-containing redox buffers were used to promote the folding of proteins that varied in properties such as size, overall tertiary structure, number of disulfide bonds, pI value, and difficulty of in vitro folding. Diselenides are able to catalyze the oxidative folding of all proteins tested, providing significant increases in both rate and yield relative to analogous disulfides. Compared to the disulfide-linked dimer of glutathione (the most commonly used oxidant for in vitro protein folding), selenoglutathione provided markedly improved efficiencies in the folding of biotechnologically important proteins such as hirudin, lysozyme, human epidermal growth factor and interferon α-2a. Selenoglutathione also enhances the renaturation of more challenging targets such as bovine serum albumin, whose native state contains 17 disulfide bonds, and the Fab fragment of an antibody. In the latter case, micromolar amounts of selenoglutathione are able to match the modest yield provided by a previously optimized redox buffer, which contains millimolar levels of glutathione. Taken together, the folding reactions of these diverse proteins exemplify the advantages and limitations of diselenide catalysts.     

Selenoglutathione: efficient oxidative protein folding by a diselenide

Diselenide bonds are intrinsically more stable than disulfide bonds. To examine how this stability difference affects reactivity, we synthesized selenoglutathione (GSeSeG), an analogue of the oxidized form of the tripeptide glutathione that contains a diselenide bond in place of the natural disulfide. The reduction potential of this diselenide bond was determined to be -407 +/- 9 mV, a value which is 151 mV lower than that of the disulfide bond in glutathione (GSSG). Thus, the diselenide bond of GSeSeG is 7 kcal/mol more stable than the disulfide bond of GSSG. Nonetheless, we found that GSeSeG can be used to oxidize cysteine residues in unfolded proteins, a process that is driven by the gain in protein conformational stability upon folding. Indeed, the folding of both ribonuclease A (RNase A) and bovine pancreatic trypsin inhibitor (BPTI) proceeded efficiently using GSeSeG as an oxidant, in the former case with a 2-fold rate increase relative to GSSG and in the latter case accelerating conversion of a stable folding intermediate to the native state. In addition, GSeSeG can also oxidize the common biological cofactor NADPH and is a good substrate for the NADPH-dependent enzyme glutathione reductase (kcat = 69 +/- 2 s-1, Km = 54 +/- 7 microM), suggesting that diselenides can efficiently interact with the cellular redox machinery. Surprisingly, the greater thermodynamic stability of diselenide bonds relative to disulfide bonds is not matched by a corresponding decrease in reactivity.     

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases.     

Oxidative protein folding: From thiol–disulfide exchange reactions to the redox poise of the endoplasmic reticulum

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI_red:PDI_ox. The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC–MS–MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment.     

Acceleration of disulfide-coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K(RCG) = 3.69 × 10(-3) s(-1)) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.     

Effect of protein and peptide inhibitors on the activity of protein disulfide isomerase

The protein disulfide isomerase catalyzed reduction of insulin by glutathione is inhibited by peptides of various length and amino acid composition. Peptide inhibitors are competitive against insulin and noncompetitive against GSH, consistent with a sequential rather than a double displacement mechanism. Peptides of unrelated primary sequence that do not contain cysteine inhibit the GSH-insulin transhydrogenase activity of PDI, and the affinity of these peptides toward the enzyme is largely dependent on the peptide length rather than composition, hydrophobicity, or charge. Cysteine-containing peptides are 4-8-fold better inhibitors than non-cysteine-containing peptides of the same length, suggesting a cysteine-specific component to the interaction with the enzyme. Oxidized insulin chain B also inhibits the oxidative folding of reduced ribonuclease in a glutathione redox buffer with an inhibition constant that is comparable to that observed for the inhibition of insulin reduction, suggesting a similar if not identical binding site for the catalysis of oxidative protein folding and the reduction of insulin.     

Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers

Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important parameter for efficient in vitro folding is the composition of the redox buffer, a mixture of a small molecule thiol and small molecule disulfide. The effects of different redox buffers on protein folding, however, have received limited attention. The oxidative folding of denatured reduced lysozyme was followed in the presence of redox buffers containing varying concentrations of five different aromatic thiols or the traditional aliphatic thiol glutathione (GSH). Aromatic thiols eliminated the lag phase at low disulfide concentrations, increased the folding rate constant up to 11-fold, and improved the yield of active protein relative to GSH. The yield of active protein was similar for four of the five aromatic thiols and for glutathione at pH 7 as well as for glutathione at pH 8.2. At pH 6 the positively charged aromatic thiol provided a higher yield than the negatively charged thiols.     

Oxidative folding of lysozyme with aromatic dithiols, and aliphatic and aromatic monothiols

In vitro protein folding of disulfide containing proteins is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. In this study, we examined redox buffers containing asymmetric dithiols 1-5, which possess an aromatic and aliphatic thiol, and symmetric dithiols 6 and 7, which possess two aromatic thiols, for their ability to fold reduced lysozyme at pH 7.0 and 8.0. Most in vivo protein folding catalysts are dithiols. When compared to glutathione and glutathione disulfide, the standard redox buffer, dithiols 1-5 improved the protein folding rates but not the yields. However, dithiols 6 and 7, and the corresponding monothiol 8 increased the folding rates 8-17 times and improved the yields 15-42% at 1mg/mL lysozyme. Moreover, aromatic dithiol 6 increased the in vitro folding yield as compared to the corresponding aromatic monothiol 8. Therefore, aromatic dithiols should be useful for protein folding, especially at high protein concentrations.     

Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin

The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the beta subunit of human chorionic gonadotropin (hCG- beta), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-beta were investigated: the rate-limiting events in the folding of this protein, and the assembly of hCG-beta with, hCG-alpha. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-beta folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10muM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the beta subunit under these conditions yields a functional protein, based on its ability to assemble with the alpha subunit. The rates of assembly of hCG-beta with hCG-alpha in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t(1/2/sb> = 9 to 12 min)). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine. (c) 1994 John Wiley & Sons, Inc.     

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.     

Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically

Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In this study, we investigated the effects of the biologically relevant thiol-disulphide redox molecule, glutathione, and Zn2+-binding on the oxidative folding of yeast mitochondrial Tim10 using both biochemical and biophysical methods in vitro. We show that, whilst oxidized Tim10 cannot be reduced by reduced glutathione, reduced Tim10 is effectively oxidized at levels of glutathione comparable to those found in the cytosol. The oxidized Tim10 generated in the presence of glutathione is competent for complex formation with its partner protein Tim9, confirming it has a native fold. The standard redox potential of Tim10 at pH 7.4 was determined to be -0.32 V, confirming that Tim10 is a much stronger reductant than glutathione (-0.26 V, at pH 7.4) and could therefore be oxidized rapidly by oxidized glutathione in the cytosol. However, we found that Zn2+-binding can stabilize the reduced Tim10, decreasing the rate of the oxidative folding more than tenfold. In addition, we show that protein disulphide isomerase can catalyse the oxidative folding of Tim10 provided that Zn2+ was removed. We propose that Zn2+-binding is essential to maintain the protein in a reduced and import-competent state in the cytosol, and that zinc has to be removed after the protein is imported into mitochondria to initiate protein oxidative folding and assembly.     

Hyperactivity of the Ero1α Oxidase Elicits Endoplasmic Reticulum Stress but No Broad Antioxidant Response

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an “oxidative environment” by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis.     

A Highlights from MBoC Selection: Kinetic control by limiting glutaredoxin amounts enables thiol oxidation in the reducing mitochondrial intermembrane space

The mitochondrial intermembrane space (IMS) harbors an oxidizing machinery that drives import and folding of small cysteine-containing proteins without targeting signals. The main component of this pathway is the oxidoreductase Mia40, which introduces disulfides into its substrates. We recently showed that the IMS glutathione pool is maintained as reducing as that of the cytosol. It thus remained unclear how equilibration of protein disulfides with the IMS glutathione pool is prevented in order to allow oxidation-driven protein import. Here we demonstrate the presence of glutaredoxins in the IMS and show that limiting amounts of these glutaredoxins provide a kinetic barrier to prevent the thermodynamically feasible reduction of Mia40 substrates by the IMS glutathione pool. Moreover, they allow Mia40 to exist in a predominantly oxidized state. Consequently, overexpression of glutaredoxin 2 in the IMS results in a more reduced Mia40 redox state and a delay in oxidative folding and mitochondrial import of different Mia40 substrates. Our findings thus indicate that carefully balanced glutaredoxin amounts in the IMS ensure efficient oxidative folding in the reducing environment of this compartment.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

Oxidative folding competes with mitochondrial import of the small Tim proteins

All small Tim proteins of the mitochondrial intermembrane space contain two conserved CX(3)C motifs, which form two intramolecular disulfide bonds essential for function, but only the cysteine-reduced, but not oxidized, proteins can be imported into mitochondria. We have shown that Tim10 can be oxidized by glutathione under cytosolic concentrations. However, it was unknown whether oxidative folding of other small Tims can occur under similar conditions and whether oxidative folding competes kinetically with mitochondrial import. In the present study, the effect of glutathione on the cysteine-redox state of Tim9 was investigated, and the standard redox potential of Tim9 was determined to be approx. -0.31 V at pH 7.4 and 25 degrees C with both the wild-type and Tim9F43W mutant proteins, using reverse-phase HPLC and fluorescence approaches. The results show that reduced Tim9 can be oxidized by glutathione under cytosolic concentrations. Next, we studied the rate of mitochondrial import and oxidative folding of Tim9 under identical conditions. The rate of import was approx. 3-fold slower than that of oxidative folding of Tim9, resulting in approx. 20% of the precursor protein being imported into an excess amount of mitochondria. A similar correlation between import and oxidative folding was obtained for Tim10. Therefore we conclude that oxidative folding and mitochondrial import are kinetically competitive processes. The efficiency of mitochondrial import of the small Tim proteins is controlled, at least partially in vitro, by the rate of oxidative folding, suggesting that a cofactor is required to stabilize the cysteine residues of the precursors from oxidation in vivo.     

Hibernation induces glutathione redox imbalance in ground squirrel intestine

Glutathione (GSH) is the major thiol-disulfide redox buffer in cells and is a critical component of antioxidant defense. Here we examined GSH redox balance in the intestinal mucosa during the annual cycle of 13-lined ground squirrels (Spermophilus tridecemlineatus). The ratio of reduced GSH to its oxidized form (glutathione disulfide, GSSG), which is an index of oxidative stress, was five-fold lower in hibernating compared with summer-active squirrels, an effect due primarily to elevated GSSG concentration in hibernators. During hibernation the total pool of GSH equivalents was lowest in squirrels undergoing arousal and highest in squirrels during interbout arousals. Hibernation decreased intestinal GSSG reductase activity by approximately 50%, but had no effect on activities of glutathione peroxidase or glucose-6-phosphate dehydrogenase. Within the hibernation season, expression of the stress protein HSP70 in intestinal mucosa was highest in squirrels entering torpor and early in a torpor bout, and lowest in squirrels arousing from torpor and during interbout euthermia. The results suggest that hibernation in ground squirrels is associated with a shift in intestinal GSH redox balance to a more oxidized state. Higher levels of HSP70 during the early phases of torpor may reflect induction of the stress response due to aberrations in protein folding or may be a mechanism to increase enterocyte tolerance to subsequent stress imposed by extended torpor or the arousal process.     

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.     

Pathway of oxidative folding of secretory leucocyte protease inhibitor: an 8-disulfide protein exhibits a unique mechanism of folding

Secretory leucocyte protease inhibitor (SLPI) is a 107-amino acid protein with a high density of disulfide pairing (eight). The mechanism of oxidative folding of reduced and denatured SLPI has been investigated here. Despite an exceedingly large number of possible folding intermediates ( approximately 46 million disulfide isomers) and their potential to complicate the refolding process, oxidative folding of SLPI turns out to be surprisingly simple and efficient. Complete oxidative folding and a near-quantitative recovery of the native SLPI can be achieved in a simple buffer solution using air oxidation without any supplementing thiol catalyst or redox agent, a phenomenon that has not yet been observed with other disulfide proteins. Because of the heterogeneity and extensive overlapping of folding intermediates, identification of the predominant intermediate was unfeasible. Nonetheless, studies of reductive unfolding of native SLPI and oxidative folding of a six-disulfide variant of SLPI enable us to propose an underlying mechanism accounting for the unique folding efficiency of SLPI in the absence of a redox agent. Our studies indicate that oxidative folding of SLPI undergoes heterogeneous populations of one-, two-, three-, four-, five-, six-, and seven-disulfide isomers, including two nativelike isomers, SLPI-6A and SLPI-7A, as transient intermediates. Formation of the last two native disulfide bonds leading to the conversion of SLPI-6A --> SLPI-7A --> N-SLPI is relatively slow and represents the final stage of oxidative folding. Most importantly, free cysteines of SLPI-6A and SLPI-7A also act as a thiol catalyst in promoting the disulfide shuffling of diverse non-native intermediates accumulated along the folding pathway. This explains why a near-quantitative recovery of N-SLPI can be achieved in the absence of any thiol catalyst and redox agent. Properties of SLPI-6A and SLPI-7A were investigated and compared to those of other documented kinetic intermediates of oxidative folding. The correlation between the mechanism of SLPI folding and the three-dimensional structure of SLPI is also elaborated.